| 体外拼装苦瓜降糖多肽P基因 |
| |
| 引用本文： | 张文姬,王碧莲,郭茸恺,赵健,王富军.体外拼装苦瓜降糖多肽P基因[J].粉末涂料与涂装,2011,24(1). |
| |
| 作者姓名： | 张文姬 王碧莲 郭茸恺 赵健 王富军 |
| |
| 作者单位： | 华东理工大学生物反应器工程国家重点实验室; |
| |
| 基金项目： | 国家十一五支撑计划(2006BAI06A162007-2009) |
| |
| 摘 要： | 目的 以苦瓜降糖多肽P基因为例,探讨用寡核苷酸片段体外拼装基因的方法。方法根据已知苦瓜降糖多肽P的蛋白序列和大肠杆菌的密码子偏爱性,反向翻译出可在大肠杆菌内表达的苦瓜降糖多肽P基因,设计24条40 bp的降糖多肽P基因寡核苷酸片段引物,通过PCR进行寡核苷酸片段的扩增和拼装。将拼装好的多肽P基因的cDNA与pET-32a载体连接后,转化E.coli BL21(DE3),IPTG诱导表达,表达产物经Ni2+-NTA纯化后检测其对四氧嘧啶引起的糖尿病小鼠的降糖活性。结果苦瓜降糖多肽P基因经2轮PCR扩增后,即可见目的 条带;经第3轮PCR扩增,即可见清晰的目的 条带。经测序鉴定,与设计的降糖多肽P基因序列完全一致。重组蛋白在E.coli BL21(DE3)中获得可溶性表达,且可与鼠抗His单抗发生特异性反应。纯化的融合蛋白纯度达90%以上,产量为10 mg/L,其对四氧嘧啶引起的糖尿病小鼠具有降糖活性。结论体外基因拼装法是一种有效获取目的 基因的方法,可进行密码子偏爱性设计,在宿主系统中有效表达目的 基因。
|
| 关 键 词： | 基因拼装 苦瓜降糖多肽P 聚合酶链反应 密码子偏爱性 |
Assembly of Momordica charantia P-polypeptide Gene In Vitro |
| |
| ZHANG Wen-ji,WANG Bi-lian,GUO Rong-kai,ZHAO Jian,WANG Fu-jun.Assembly of Momordica charantia P-polypeptide Gene In Vitro[J].Chinese Journal of Biologicals,2011,24(1). |
| |
| Authors: | ZHANG Wen-ji WANG Bi-lian GUO Rong-kai ZHAO Jian WANG Fu-jun |
| |
| Affiliation: | ZHANG Wen-ji,WANG Bi-lian,GUO Rong-kai,ZHAO Jian,WANG Fu-jun(State Key Laboratory of Bioreac-tor Engineering,East China University of Science and Technology,Shanghai 200237,China) |
| |
| Abstract: | Objective To investigate the method for in vitro gene assembly using oligonucleotide(oligos)using Momordica charantia P-polypeptide gene as an example.Methods The Momordica charantia P-polypeptide gene which might be expressed in E.coli was reversely translated according to the known protein sequence of the said gene and the codon-preference of E.coli,based on which 24 primers for oligos fragments of Momordica charantia P-polypeptide gene,each at a length of 40 bp,were designed for amplification and assembl... |
| |
| Keywords: | Gene assembly Momordica charantia P-polypeptide Polymerase chain reaction Codon preference |
| 本文献已被 CNKI 万方数据 等数据库收录！ |
|
